CN113943686B - Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain - Google Patents
Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain Download PDFInfo
- Publication number
- CN113943686B CN113943686B CN202111439195.6A CN202111439195A CN113943686B CN 113943686 B CN113943686 B CN 113943686B CN 202111439195 A CN202111439195 A CN 202111439195A CN 113943686 B CN113943686 B CN 113943686B
- Authority
- CN
- China
- Prior art keywords
- culture
- pasteurella multocida
- strain
- culture medium
- percent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 71
- 241000606856 Pasteurella multocida Species 0.000 title claims abstract description 60
- 229940051027 pasteurella multocida Drugs 0.000 title claims abstract description 60
- 238000012136 culture method Methods 0.000 title abstract description 7
- 241000282898 Sus scrofa Species 0.000 claims abstract description 36
- 238000000855 fermentation Methods 0.000 claims abstract description 35
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 239000000843 powder Substances 0.000 claims abstract description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 239000001888 Peptone Substances 0.000 claims abstract description 11
- 108010080698 Peptones Proteins 0.000 claims abstract description 11
- 235000015278 beef Nutrition 0.000 claims abstract description 11
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 11
- 239000000284 extract Substances 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 11
- 235000019319 peptone Nutrition 0.000 claims abstract description 11
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 239000012138 yeast extract Substances 0.000 claims abstract description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 19
- 241001465754 Metazoa Species 0.000 claims description 14
- 241000282887 Suidae Species 0.000 claims description 13
- 230000001954 sterilising effect Effects 0.000 claims description 13
- 210000004369 blood Anatomy 0.000 claims description 12
- 239000008280 blood Substances 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 9
- 238000005273 aeration Methods 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 8
- 239000013530 defoamer Substances 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 abstract description 2
- 229960005486 vaccine Drugs 0.000 description 32
- 241000283973 Oryctolagus cuniculus Species 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 13
- 238000004108 freeze drying Methods 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 238000002156 mixing Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 10
- 239000003708 ampul Substances 0.000 description 10
- 239000006161 blood agar Substances 0.000 description 10
- 238000007689 inspection Methods 0.000 description 10
- 239000008215 water for injection Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000003223 protective agent Substances 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000002518 antifoaming agent Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 238000013207 serial dilution Methods 0.000 description 5
- 206010034107 Pasteurella infections Diseases 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 201000005115 pasteurellosis Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 206010035664 Pneumonia Diseases 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000002498 deadly effect Effects 0.000 description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 235000001727 glucose Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 235000014347 soups Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 201000008197 Laryngitis Diseases 0.000 description 1
- 208000032912 Local swelling Diseases 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010034829 Pharyngeal oedema Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000005831 deiodination reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 201000006509 pleuropneumonia Diseases 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain, wherein the culture medium comprises the following components in percentage by mass: 2 to 3 percent of yeast extract powder, 2 to 3 percent of beef extract powder, 1 to 1.5 percent of peptone, 0.6 to 0.8 percent of glucose, 0.1 to 0.3 percent of sodium chloride, 0.2 to 0.5 percent of potassium dihydrogen phosphate, 0.5 to 0.8 percent of disodium hydrogen phosphate and the balance of water. The culture medium of the swine pasteurella multocida can realize ventilation fermentation culture of the swine pasteurella multocida under a proper environment, the fermentation culture time is short (only 14-16 h is needed), the viable count can reach 170-200 multiplied by 10 8 CFU/ml, and the culture medium has excellent industrial application value.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain.
Background
The swine pasteurellosis (Swine pasteurellosis) is a sporadic infectious disease of swine caused by Pasteurella multocida, also called swine plague, laryngeal wind or "neck swelling disease", and mainly causes symptoms such as swine throat swelling, dyspnea, acute septicemia, pneumonia and the like. The disease is a common infectious disease of pigs, can occur all the year round, is parasitic to mice and other rodents, has pathogenicity to various animals and people, is most easy to infect pigs, has no obvious seasonal occurrence, but is more in wet and rainy condition, and causes such as malnutrition, long-distance transportation, change of feeding conditions, bad disease and the like to promote the occurrence of the disease, and is frequently and intensively developed. It is characterized in that most acute sepsis and pharyngitis are caused by laryngitis; acute type is cellulose pleuropneumonia; chronic forms are less common and mainly manifest chronic pneumonia.
The pasteurellosis vaccine plays an important role in treating swine pasteurellosis. The live bacteria for growing the pasteurella multocida on a large scale are key for preparing high-quality vaccines. At present, martin broth, modified Martin broth culture and the like are generally used for culturing Pasteurella multocida. The preparation process of the culture mediums is complex, comprises the procedures of meat purchasing, cold storage preservation, picking, mincing, heat preservation digestion, extraction and filtration and the like, and needs to consume a great deal of manpower and material resources, has a longer production period, is influenced by the quality of meat, and influences the stability of the quality of the culture mediums, thereby influencing the quality of vaccines. The reproductive capacity of the pasteurella multocida in pigs is related to the bacterial strain and the composition of a culture medium, and is also related to the culture conditions such as the culture temperature, the pH value, the culture time and the like. Therefore, the research on the growth and propagation culture medium suitable for the swine pasteurella multocida and the culture method thereof has very important practical significance.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain, wherein the culture medium has short culture time and can promote high-yield viable count of the swine pasteurella multocida.
In order to achieve the aim, the invention provides a culture medium of a swine pasteurella multocida EO630 strain, which comprises the following components in percentage by mass: 2 to 3 percent of yeast extract powder, 2 to 3 percent of beef extract powder, 1 to 1.5 percent of peptone, 0.6 to 0.8 percent of glucose, 0.1 to 0.3 percent of sodium chloride, 0.2 to 0.5 percent of potassium dihydrogen phosphate, 0.5 to 0.8 percent of disodium hydrogen phosphate and the balance of water.
The preparation method of the culture medium comprises the following steps: weighing the components with the required amount according to the culture medium composition, uniformly mixing, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to fix the volume, and sterilizing at 116 ℃ for 40min to obtain the culture medium.
The culture medium contains commercial biochemical reagents such as beef extract powder, yeast extract powder, peptone, glucose, sodium chloride, potassium dihydrogen phosphate and the like, has the advantages of convenient acquisition of culture medium components, simple and convenient process, stable quality, short production period, low manufacturing cost and the like.
Preferably, the medium comprises the following components in mass percent: 2.2 to 2.7 percent of yeast extract powder, 2.3 to 2.6 percent of beef extract powder, 1.2 to 1.4 percent of peptone, 0.7 to 0.75 percent of glucose, 0.15 to 0.25 percent of sodium chloride, 0.3 to 0.4 percent of potassium dihydrogen phosphate, 0.6 to 0.7 percent of disodium hydrogen phosphate and the balance of water.
The second aspect of the present invention provides a method for culturing a pasteurella multocida EO630 strain, the method comprising the steps of:
S1, diluting and inoculating a pasteurella multocida EO630 strain to a flat plate containing 0.1-0.2% of cracked whole blood and 3-5% of healthy animal serum, and culturing for 16-24 hours at 35-37 ℃ to obtain first-stage seeds of the pasteurella multocida EO630 strain; the plate is Martin agar plate;
S2, inoculating the first-level seeds of the porcine pasteurella multocida EO630 strain into a culture medium, and culturing for 18-20 hours at 36-37 ℃ to obtain the second-level seeds of the porcine pasteurella multocida EO630 strain;
S3, adding a culture medium into a fermentation tank according to 70-75 v/v percent, adding 0.1-0.2 v/v percent of defoamer according to the volume of the culture medium, sterilizing, reducing the temperature of the culture medium to 30-35 ℃, inoculating secondary seeds of the swine pasteurella multocida according to the inoculum size of 1-3 v/v percent, and culturing for 14-16 h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida.
Wherein the culture medium is the culture medium.
The culture medium is used for realizing ventilation fermentation culture of the swine pasteurella multocida under a proper environment, and compared with the modified Martin soup culture medium (the culture time is 16-20 h), the fermentation culture time is short (only 14-16 h is needed), the viable count of the swine pasteurella multocida can reach 170-200 multiplied by 10 8 CFU/ml, and the swine pasteurella multocida has excellent industrial application value.
Preferably, in step S3, the pH of the medium is 7.0 to 7.8.
Preferably, in step S3, the cultivation is performed by a aeration cultivation method.
The third aspect of the invention provides the use of a fermentation culture obtained by culturing by the culture method described above for the preparation of a medicament or vaccine.
The pasteurella multocida liquid of the invention is prepared into vaccine, and can be used for preventing and treating related diseases caused by the pasteurella multocida.
Specifically, the fermentation culture is placed in a sealed manner at the temperature of 2-8 ℃ for 3-7 days for use.
And more specifically, the freeze-drying protective agent and the zymophyte liquid are mixed according to the proportion of 1 (6-8) and then freeze-dried to obtain the microbial inoculum.
Further, the freeze-drying protective agent is a mixture of gelatin and sucrose, and after mixing, the mass fraction of the gelatin is 1-2%, and the mass fraction of the sucrose is 4-6%.
Through the technical scheme, the invention has the following beneficial effects:
1. The invention improves the culture medium composition of the swine pasteurella multocida, and the culture medium contains commercial biochemical reagents such as beef extract powder, yeast extract powder, peptone, glucose, sodium chloride, potassium dihydrogen phosphate and the like, and has the advantages of convenient acquisition of the culture medium composition, simple and convenient process, stable quality, short production period, low manufacturing cost and the like.
2. The culture medium of the Pasteurella multocida can realize the aeration fermentation culture of the Pasteurella multocida in a proper environment, has short fermentation culture time (only 14-16 h is needed) compared with the culture medium of the improved Martin soup (the culture time is 16-20 h), has the viable count of 170-200 multiplied by 10 8 CFU/ml, and has excellent industrial application value.
3. The pasteurella multocida liquid can be prepared into vaccine, and can be used for preventing related diseases caused by the pasteurella multocida.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to examples. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
In the following examples, beef extract powder (LAB-LEMCO POWDER) was purchased from Thermo FISHER SCIENTIFIC, england under the trade designation LP0129; yeast extract powder was purchased from Hubei Angel Yeast Co., ltd., product number FM888; peptone was purchased from Hubei Angel Yeast Co., ltd., product number FP328; glucose was purchased from Shanghai national pharmaceutical Congress chemical company, cat# 10010518; sodium chloride is available from Shanghai national pharmaceutical group chemical company, inc., cat# 10019318; monopotassium phosphate is purchased from Shanghai national pharmaceutical group chemical reagent Co., ltd, cat# 10017518; disodium hydrogen phosphate dodecahydrate was purchased from Shanghai national pharmaceutical group chemical reagent Co., ltd, cat# 10010318; modified Martin broth dry powder media was purchased from Beijing aobo Co., ltd. Biotechnology, lot number 201816; the pasteurella multocida EO630 strain was identified, stored and supplied by the hapharmaceutical group biological vaccine limited.
Example 1
S1, uniformly mixing 2g of yeast extract powder, 2g of beef extract powder, 1g of peptone, 0.6g of glucose, 0.1g of sodium chloride, 0.2g of monopotassium phosphate and 0.5g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 16 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.1% of lysed whole blood and 3% of healthy animal serum, and culturing for 16 hours at 35-37 ℃.8 colonies with the same medium size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 16 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being tested to be pure qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium prepared in step S1) for culturing for 18h at 36-37 ℃, and obtaining the second seed of the swine Pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 70% (v/v) of the volume of a fermentation tank, adding 0.1% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 1% (v/v), conducting aeration culture, and culturing for 14h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 3 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 6:1, and freeze-drying to obtain the vaccine.
Example 2
S1, uniformly mixing 3g of yeast extract powder, 3g of beef extract powder, 1.5g of peptone, 0.8g of glucose, 0.3g of sodium chloride, 0.5g of monopotassium phosphate and 0.8g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 24 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 24 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 24 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 35 ℃, inoculating the second-level seeds according to the inoculation amount of 3% (v/v), conducting aeration culture, and conducting culture for 16h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 7 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Example 3
S1, uniformly mixing 2.2g of yeast extract powder, 2.3g of beef extract powder, 1.2g of peptone, 0.7g of glucose, 0.15g of sodium chloride, 0.3g of potassium dihydrogen phosphate and 0.6g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 18 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.1% of lysed whole blood and 4% of healthy animal serum, and culturing for 18 hours at 35-37 ℃. 9 colonies with medium size and uniform size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 18 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being tested to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 19h at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 70% (v/v) of the volume of a fermentation tank, adding 0.1% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing the culture medium by high-pressure steam at 116 ℃ for 40min, reducing the temperature of the culture medium to 33 ℃, inoculating the second-level seeds according to the inoculation amount of 1-3% (v/v), conducting aeration culture, and culturing for 15h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 5 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 7:1, and freeze-drying to obtain the vaccine.
Example 4
S1, uniformly mixing 2.7g of yeast extract powder, 2.6g of beef extract powder, 1.4g of peptone, 0.75g of glucose, 0.25g of sodium chloride, 0.4g of potassium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 20 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 20 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 20 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 2% (v/v), conducting aeration culture, and conducting culture for 16h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 6 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Comparative example
S1, heating purified water to more than 80 ℃, weighing 100g of culture medium dry powder according to the preparation instructions of a purchased finished culture medium modified Martin broth dry powder culture medium (Beijing ao Boxing Biotechnology Co., ltd.), pouring the culture medium dry powder into a culture medium preparation barrel, heating the water to melt the culture medium dry powder, continuously stirring the culture medium dry powder in the water adding process, adding the water to 100ml, and properly prolonging the stirring time according to the melting degree of the culture medium dry powder until the culture medium dry powder is completely melted.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 20 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 20 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 20 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (3500 ml of S1 culture medium is filled) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine Pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 48 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 2% (v/v), conducting aeration culture, and conducting culture for 20h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 6 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Performance detection
1. Detection of viable count of fermentation culture
The inspection method comprises the following steps: the fermentation cultures prepared in examples 1-4 and comparative example were counted in three annexes of the pharmacopoeia of the animal of the people's republic of China. Colony calculation and result judgment: and (3) visually observing the bacterial colonies, counting the number of the bacterial colonies on the bottom surfaces of the flat plates, and calculating the average bacterial colony number of the 3 flat plates, namely the total bacterial colony number contained in each milliliter of stock solution. If the colony grows in a sheet shape or the colony between different plates of the same dilution is different by more than 50%, the colony should be rechecked.
The number of viable bacteria in the fermentation cultures of the Pasteurella multocida pigs produced in examples 1 to 4 and comparative example, respectively, were 170×108CFU/ml、200×108CFU/ml、183×108CFU/ml、191×108CFU/ml、82×108CFU/ml.
2. Vaccine safety test
25 Healthy rabbits weighing 1.5-2kg are used, each group is divided into 5 groups, 5 vaccine 3 bottles of each group of the vaccine of the example and the comparative example are respectively taken and mixed, the vaccine is diluted into 10 parts per ml according to the parts of the bottle label, the skin (such as the inner side of thighs) of the injection site of the test rabbits is sterilized by 3% iodine, the iodine is removed by 75% alcohol cotton, and 1.0ml of the vaccine is injected into each rabbit by a 2.0ml syringe in a subcutaneous mode. The rabbits injected with the vaccines of examples 1 to 4 were alive and the rabbits injected with the vaccine of the comparative example were dead 1 after 10 days of observation.
3. Vaccine efficacy test
Efficacy test: either of the following methods is optional.
Test with mice: 16-18 g mice were 86 in total, and divided into 5 groups of 16 mice each, with 6 controls. Operation method and result judgment: the vaccine samples 1 bottle of example 1-example 4 and comparative example were taken, the vaccine was diluted to 0.2ml (1/30 head parts) in the bottle label, the skin of the injection site of the test mice (10) was sterilized with 75% alcohol cotton balls, and each subcutaneous injection was 0.2ml. After 14 days, 3 control mice under the same conditions were injected subcutaneously with 2MLD of Pasteurella multocida C44-8 strain (CVCC 44408) and 1MLD was injected subcutaneously with 3 control mice, and the calculation method for challenge was found in the standard procedure for bacterial preparation efficacy test attack deadly trap. The mice challenged with 2MLD were all killed, 2 mice challenged with 1MLD were killed, and 5 mice vaccinated with the vaccines of examples 1-4 survived with the vaccine of comparative example.
Test with rabbits: 1.5-2 kg rabbits are divided into 5 groups of 4 rabbits and 2 rabbits are compared, and the mass standard accords with the animal standards for production inspection of the animal pharmacopoeia of the people's republic of China. The rabbit raising management is carried out according to the experimental rabbit raising management program. Operation method and result judgment: sample 1 bottle was taken, the vaccine was diluted to 1/3 head/ml according to the bottle label, the skin of the injection site of the test rabbit (such as the inner thigh) was sterilized with 3% iodine, and the vaccine of example 1-example 4 and comparative example was injected into each group of rabbits, respectively, 1.0ml each, by deiodination with alcohol cotton. After 14 days of inoculation, 2 rabbits are inoculated together with the same condition, and each is subcutaneously injected with 80-100 CFU live bacteria of pasteurella multocida C44-8 strain (CVCC 44408), and the calculation method for the challenge is shown in the standard operation procedure of bacterial product efficacy test attack deadly trap. The control rabbits should all die after 10 days of observation, the immunized rabbits of the vaccines of examples 1-4 all survive, and 2 rabbits die after the vaccine of the comparative example is injected.
Inspection with pigs: healthy and susceptible pigs 28, which are about 20kg in weight after weaning for 1 month, are divided into 5 groups of 5 pigs each, and the other 2 pigs are used as controls. The animal quality standard accords with the animal standard for production inspection in the animal pharmacopoeia of the people's republic of China, and the pig raising management is carried out according to the inspection pig raising management program. Taking 1 bottle of vaccine sample of example 1-example 4 and comparative example, diluting the vaccine to 1 part/ml according to the bottle label, sterilizing the skin of the injection site of the test pig (such as the neck behind the ear) with 3% iodine, injecting 1.0ml subcutaneously each, inoculating 14 days later, and injecting 1MLD of pasteurella multocida C44-1 (CVCC 44401) virulent bacteria liquid subcutaneously in each pig together with 3 heads of the control pigs under the same condition, wherein the method for calculating the toxin is shown in the standard operation procedure of the test for efficacy of bacterial products, deadly trap. When the control pigs all died, the immunized pigs injected with the vaccines of examples 1 to 4 all survived, and the immunized pigs injected with the vaccines of the comparative examples died 1 head.
The preferred embodiments of the present invention have been described in detail above with reference to the examples, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (4)
1. The culture medium for the pasteurella multocida EO630 strain is characterized by comprising the following components in percentage by mass: 2.2-2.7% of yeast extract powder, 2.3-2.6% of beef extract powder, 1.2-1.4% of peptone, 0.7-0.75% of glucose, 0.15-0.25% of sodium chloride, 0.3-0.4% of potassium dihydrogen phosphate, 0.6-0.7% of disodium hydrogen phosphate and the balance of water.
2. A method for culturing a pasteurella multocida EO630 strain of pigs, comprising the steps of:
S1, diluting and inoculating a pasteurella multocida EO630 strain to a flat plate containing 0.1-0.2% of cracked whole blood and 3-5% of healthy animal serum, and culturing at 35-37 ℃ for 16-24 hours to obtain first-stage seeds of the pasteurella multocida EO630 strain;
s2, inoculating the primary seeds of the porcine pasteurella multocida EO630 strain into a culture medium, and culturing at 36-37 ℃ for 18-20 hours to obtain the secondary seeds of the porcine pasteurella multocida EO630 strain;
S3, adding a culture medium into a fermentation tank according to 70-75 v/v%, adding 0.1-0.2 v/v% of defoamer based on the volume of the culture medium, sterilizing, reducing the temperature of the culture medium to 30-35 ℃, inoculating secondary seeds of the swine pasteurella multocida EO630 strain according to the inoculum size of 1-3 v/v%, and culturing for 14-16 h at 36-37 ℃ and pH value of 7.2-7.5 to obtain a fermentation culture of the swine pasteurella multocida EO630 strain;
wherein the medium is the medium according to claim 1.
3. The method of claim 2, wherein in step S3, the pH of the culture medium is 7.0 to 7.8.
4. The method of culturing a strain of pasteurella multocida EO630 according to claim 2, characterized in that in step S3, the culture is carried out by aeration culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111439195.6A CN113943686B (en) | 2021-11-30 | 2021-11-30 | Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111439195.6A CN113943686B (en) | 2021-11-30 | 2021-11-30 | Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113943686A CN113943686A (en) | 2022-01-18 |
| CN113943686B true CN113943686B (en) | 2024-05-14 |
Family
ID=79339067
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111439195.6A Active CN113943686B (en) | 2021-11-30 | 2021-11-30 | Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113943686B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111040971A (en) * | 2019-12-30 | 2020-04-21 | 青岛高科技工业园海博生物技术有限公司 | Enrichment culture medium for swine plague vaccine |
-
2021
- 2021-11-30 CN CN202111439195.6A patent/CN113943686B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111040971A (en) * | 2019-12-30 | 2020-04-21 | 青岛高科技工业园海博生物技术有限公司 | Enrichment culture medium for swine plague vaccine |
Non-Patent Citations (3)
| Title |
|---|
| 以23号培养基制造猪肺疫氢氧化铝菌苗的研究;夏君明;中国兽医科技(12);第12页第（一）材料依据和培养基命名部分，第13-14页第（三）试验结果部分 * |
| 猪多杀性巴氏杆菌病活疫苗（EO630株）生产用合成培养基的研究;崔水保等人;中国兽药杂志;第48卷(第5期);摘要，第22页右栏实验方法部分，第24页左栏第3段 * |
| 猪肺疫EO-630加糖培养增菌试验;王洪图;夏福库;;中国预防兽医学报(05);第4页左栏第2段、右栏第3段，第5页表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113943686A (en) | 2022-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108342434B (en) | Veterinary clostridium putrefaction toxin, preparation method thereof and special culture medium | |
| CN108904796B (en) | Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof | |
| CN107569681B (en) | Bivalent inactivated vaccine for bovine pasteurellosis and preparation method thereof | |
| Gall et al. | The isolation and preliminary study of some physiological characteristics of the predominating flora from the rumen of cattle and sheep | |
| CN107299070B (en) | D-type clostridium perfringens toxin for livestock and preparation method and special culture medium thereof | |
| CN108949619A (en) | A kind of zymotechnique of riemerella anatipestifer | |
| CN110201153B (en) | Triple inactivated vaccine for rabbit viral hemorrhagic disease, pasteurellosis and bordetella disease and preparation method thereof | |
| CN101926987A (en) | Method for producing piglet paratyphoid live vaccine by using synthetic medium | |
| CN113943686B (en) | Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain | |
| CN112574924A (en) | Bacillus subtilis strain, microecological preparation and application thereof | |
| CN109554420B (en) | Clostridium perfringens type B exotoxin and preparation method, toxin production medium and application thereof | |
| CN109609418A (en) | One plant of erysipelothrix porci and its application | |
| CN111040971A (en) | Enrichment culture medium for swine plague vaccine | |
| CN113862196B (en) | Bacillus subtilis SD-KC-001 and application thereof | |
| CN109010814A (en) | The production method of haemophilus parasuis and mycoplasma hyopneumoniae bivalent inactivated vaccine | |
| CN111514285B (en) | Mycoplasma ovipneumoniae, type-A pasteurella multocida and type-D pasteurella multocida triple inactivated vaccine | |
| CN105420153B (en) | A kind of Pseudomonas aeruginosa fermentation medium and its fermentation culture method, vaccine preparation method | |
| CN104740622B (en) | Pseudomonas aeruginosa, klebsiella and pasteurella triple-inactivated vaccine for mink | |
| CN107496913A (en) | Brickpox, pig parvoviral, swine influenza virus triple inactivated vaccine preparation method | |
| CN101648013A (en) | Preparation method of inactivated vaccine of infectious coryza of chicken | |
| CN111548965A (en) | Donkey-derived bacillus pumilus and application thereof in preparation of medicine for treating diarrhea of donkey colt | |
| CN104645324A (en) | Application of swine enzootic hyopneumoniae vaccine strain | |
| CN116983419B (en) | Universal heat-resistant freeze-drying protective agent and preparation method thereof | |
| CN116445373B (en) | C clostridium perfringens toxigenic culture medium and preparation method and application thereof | |
| CN112107681B (en) | Triple four-prevention inactivated vaccine for fast plague, sudden sniper, lamb dysentery and enterotoxemia and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |